Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Thank you for the summary of our paper.Let us correct one point: this paper proposes dot blotting as a method that does not require desorption of antigens.Therefore, we consider that quantitative determination of desorption is unnecessary.
On the other hand, as you pointed out, each experiment was not repeated, and statistical analyses were lacking.This time, we conducted additional experiments, performed statistical analyses on each experimental result, and made corrections.Would you confirm the revised Results section?
Antigen that is adsorbed to the alum matrix is not accessible to immune detection by dot blotting, but is immunologically active.If the assay is meant to measure the antigen content IT IS NECESSARY to quantitatively determine the amount of antigen.If the authors refuse to provide evidence for this, the paper is not suited for publication.
Showing an error bar is no statistical analysis.
Why has the comment regarding the broad body of literature been ignored? 3 Line 86 ff.-adsorption of PMT on aluminium hydroxide -The process of alum adsorption is relatively fast, but the duration of the adsorption process has an influence on the strength of binding.An over night adsorption might not be representative for commercial products.
As you pointed out, it is possible that the adsorption of the antigen onto the aluminum gel is completed without waiting overnight.The overnight reaction step is thought to be due to efficiencies in the vaccine manufacturing process.In this paper, we prepared each sample according to the requirements for vaccine approval and aimed to quantify the amount of antigen in the vaccine.The reviewer's comment has not been answered.The adsorption process continues during storage.An overnight adsorption is too short.
4 Usually, antigens -such as tetanus toxoids-are formalin treated before alum adsorption.What is the rationale for the fixation step after the adsorption?
As you pointed out, toxoids are usually "inactivated'' with formalin beforehand because the antigenic toxins have pathogenicity.However, as we answered to Editor's comment No.2, the antigens of the AR and AR/ER vaccines discussed in this paper are recombinant non-toxic mutant toxins, so an "inactivation" step is unnecessary.The main purpose of formalin treatment after aluminum gel adsorption is "fixation."In fact, the need for this "fixation" treatment is not scientifically justified, but the samples were prepared in a manner similar to vaccine production, in accordance with veterinary drug approvals.
It is not convincing to read that there was no scientific rationale for the fixation treatment.For vaccine production formalin fixation is done before alum adsorption.The process described by the authors is not comparable.
5 Line 101 -Neutralizing activity of anti PMT MAb: Please provide a reference.
Unfortunately, there are no published papers on the neutralizing titers of this antibody.As a result of inhouse testing in the past following the method described on page 8, line 128, it was confirmed that this MAb has 256 times more neutralizing capacity.
We have added the neutralizing antibody titers on page 6, line 104.
Line 108 is a head line, monoclonal antibodies are not mentioned.

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Line 106 -Dot blotting -Desorption of antigens from aluminum hydroxide salts is achieved by high ionic strength and/or high pH of the desorption buffer (as alum reaches neutral charge at ~pH 9).In addition, the process is time dependent.The process described in the manuscript uses neither of these mechanisms.It is hard to conceive that quantitative desorption was reached.This should be demonstrated, also with commercial vaccine batches.
The dot blotting method described in this paper is proposed as a direct quantitative method for the antigen-adjuvant complex that does not require the desorption of the antigen from the aluminum gel.Therefore, we believe that treatment with high ionic strength or high pH is unnecessary in this test system.
The personal conviction of the authors is irrelevant.The question was, whether ER interferes with the quantitative detection of PMT antigens, e.g. by reducing the binding capacity of the nitrocellulose membrane.This question was not addressed.
If this is a PMT-specific mAb, why is there reactivity with ER antigens?.

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-What is the 95% confidence interval of the antibody titres?Are the titres significantly different?
Statistical analysis of neutralizing antibody titers has been added to Table S4.The difference in geometric mean values were observed, but no statistically significant differences were observed due to individual dispersion.
What do the authors conclude from the figure, if the data is not significant and does not show any trend?
As you pointed out in No. 2 and No. 7, the error was due to the lack of the number of replicates of the experiment.Multiple experiments have been repeated and it has been shown that the amount of free antigen in the supernatant is dependent on the concentration of aluminum gel.We would appreciate your confirmation of the revised Fig 2.